PROJECT SUMMARY: In 2016, the U.S. Food and Drug Administration warned that repeated or lengthy use of general anesthetics in children below the age of three might affect their brain development. This warning raises serious concerns regarding the safety of pediatric anesthesia. There are two main barriers in the research field of anesthetic-induced developmental neurotoxicity (AIDN): 1) So far, most of the evidence for AIDN was obtained from animal studies. The results from human studies remain inconclusive. 2) The mechanisms are largely unknown. The goal of this proposed study is to address both of these barriers. First, we established a new in vitro system of three-dimensional (3D) human mini brains using induced pluripotent stem cells for modeling human brain development. Human mini brains are more similar to developing human brains, both structurally and functionally, than the widely used 2D neurons. Thus, application of human mini brains in AIDN research field helps bridge the gap between the animal and human studies. Our preliminary data provided the first evidence showing that clinically relevant doses of either propofol or sevoflurane, two commonly used pediatric anesthetics, induced cell death in human mini brains. Second, we recently used an unbiased approach to screen the expression of 24,881 long non-coding RNAs (lncRNAs) and 35,923 messenger RNAs in neonatal mouse hippocampi. We discovered that the expression levels of the lncRNA AK156531 gene, and its nearby protein-coding gene Neuronal Per Arnt Sim domain protein 4 (NPAS4), were dramatically decreased following propofol exposure. One of the known functions of lncRNAs is to regulate their nearby gene expression. We found that knockdown of AK156531 decreased NPAS4 levels in both human mini brains and mouse brains, strongly suggesting that AK156531 might regulate NPAS4 expression. NPAS4 is involved in excitatory/inhibitory (E/I) balance, learning and memory, and neuroprotection. We also found that neonatal propofol exposure caused multiple adverse effects in mice (E/I imbalance, neuronal death, and impaired memory function). These exciting findings, combined with the reported function of NPAS4, suggest that the abnormally expressed AK156531 might directly contribute to AIDN. Thus, we propose to utilize AK156531 gain- and loss-of-function approaches to examine the role and mechanism of AK156531 in AIDN in mice, and to facilitate the translation of these findings to humans by using human mini brains. We hypothesize that downregulation of AK156531 contributes to E/I imbalance, neuronal death and cognitive dysfunction via NPAS4 signaling. For the first time in this field, human mini brains will be combined with AK156531 knockdown and overexpression mouse models to investigate the novel mechanisms of lncRNA involvement. This proposal is expected to provide new mechanistic insights into the neurodevelopmental consequences of pediatric anesthetic exposure. This will further aid in the development of more rational neuroprotective strategies related to pediatric anesthetic use, and movement towards a better assurance of safety for pediatric anesthesia use.